Burn is any extremity experienced by the skin caused by heat, cold, electricity, chemicals, friction or radiation. Burn injury is generally associated with tissue fluid and electrolyte imbalance, decelerated healing of the burnt area, metabolic disturbance, muscle catabolism and various other complications of vital organs and may further have secondary complications like infection by bacteria and/or micro-organisms. Each of these complications needs different agents/drugs to provide a comprehensive burn management in saving the life.
Burns related injuries and death is a major problem in South East Asia, especially in developing countries like India. As per WHO reports. South East Asia region contributes towards 10% of global burden, with India alone accounting for 35,000 burn related deaths annually. Even in a developed country such as USA, an estimated 2.1 million Americans seek medical treatment each year for burns. One of the major causes of mortality in burn patients is severe systemic infection. The primary route of entry of these infections is the exposure of raw burn surface to various bacteria and pathogens. Factors, such as disruption of the skin barrier, a large cutaneous bacterial load, the possibility of the normal bacterial flora turning into opportunistic pathogens and the severe depression of the immune system contribute towards sepsis in a burns victim, which usually is life threatening [1]. Appropriate wound management of burn surface and early re-epitheliazation with wound closure is a vital step in the treatment and prognosis of burn patients.
Infection control is a very important process in the prevention of secondary infections and also in maintaining a proper burn wound healing process. Although the use of topical antimicrobial agents is essential in the establishment of the bacterial balance in burn wounds, it has been associated with delayed healing of burn wounds in which the process of skin proliferation and collagen deposition play a major role [2]. Some studies showed that the wound healing process is delayed, which is significant with second and third degree burns, due to delayed or less production of growth promoting factors like Recombinant Human Epidermal Growth Factor (rh-EGF) or Platelet Derived Growth Factor (rh-PDGF-BB) using their receptors.
Recombinant Human Epidermal Growth Factor rh-EGF belongs to a family of growth factors that regulate cell proliferation, migration and differentiation through binding to receptor kinase on target cells [3]. rh-EGF has been shown to act as a potent mitogen and also as a differentiation factor for many cell types including smooth muscle cells [4]. Experimental studies in animals have demonstrated that topical application of rh-EGF accelerates the rate of epidermal regeneration of partial thickness wounds and second degree burns [5]. rh-EGF is 53 amino acids protein with a molecular weight of 6.2 kDa and is obtained by recombinant gene technology. A novel vector was constructed encoding synthetic rh-EGF polynucleotide sequence, which was over-expressed in E. coli and purified to obtain >98% pure protein [6].
In the process of wound healing the signal for cellular proliferation is given by rh-EGF peptide via EGF receptors. The EGF receptors have tyrosine kinase transmembrane domains, with a cytoplasmic domain and extra cellular domain, which are involved in rh-EGF binding. This results in EGF receptor dimerization, autophosphorylation of the receptor and tyrosine phosphorylation of other proteins. This activates mitogen activated protein kinase (MAP kinase) pathway, ultimately causing phosphorylation of transcription factors such as C-Fs to create AP-1 and ELK-1 that contribute to proliferation. Activation of STAT-1 and STAT-3 transcription factors by JAK kinase in response to rh-EGF contribute to proliferative signalling. Further, phosphatidylinositol signalling and calcium release induced by rh-EGF active protein kinase C is another component of EGF signalling. The above process attracts cells into wounds and stimulates their proliferation, enhances the rate of formation of granulation tissue and increase collagen production [7, 8].
Though the growth factors like rh-EGF help in healing the burn wounds, it is important to prevent the infection caused during burn wound management. Recombinant human epidermal growth factor rh-EGF (REGEN-D, Bharat Biotech International Limited), which was cloned and over expressed in E. coli, has shown enhanced healing of burn wounds by significantly reducing the duration of healing. But the risk associated with burn wounds is invasion of infection by micro-organisms. This is where the antimicrobial agents play significant role in burn wound management.
In the prior art, various topical antibacterial/antimicrobial agents are available for wound care like Bacitracin, Polymyxin B sulfate, Neomycin, Povidone-iodine, Mafenide acetate cream, Nitrofurazone, Gentamicin etc.
Bacitracin is a polypeptide antibiotic that is effective against Gram-positive cocci and bacilli. Bacitracin may also enhance re-epithelialization of the wound, though it has no affect on keratinocyte proliferation. Incidence of resistant strains is unlikely to increase because bacitracin acts on the properties of the bacterial plasma membrane and not on molecular synthesis [9].
Polymyxin B sulfate is a simple basic peptide antibiotic that is effective against Gram-negative organisms. Polymyxin B sulphate causes a greater reduction of keratinocyte proliferation [10].
Neomycin is broad-spectrum antibiotic and is particularly effective against Gram-negative organisms. However, side effects like hypersensitivity reactions, particularly skin rashes occur more frequently with neomycin [11].
Povidone-iodine is a bactericidal effective against Gram-positive and Gram-negative bacteria. Povidone-iodine at clinical concentration has been shown to be toxic to human fibroblasts and keratinocytes in vitro. Povidine-iodine has also been reported to be inactivated by wound exudates. This topical agent may harden wound eschar rather than soften it, thus increasing the difficulty and discomfort of wound debridement [1].
Mafenide acetate is a methylated topical sulfonamide compound. This drug has wide range of antibacterial activity against most Gram-negative and Gram-positive pathogens. However, use of mafenide may be inhibitory to re-epithelialization. Mafenide suppresses Polymorphonuclear Leukocytes (PMN) and lymphocyte activity [16].
Nitrofurazone compound is abroad spectrum antibacterial effective against S. aureus, Enterobactor and E. coli, but it is less effective against P. aeruginosa and has no significant fungicidal activity. Nitrofurazone has been shown to have a detrimental effect on the growth and migration of keratinocytes in culture [17]. Nitrofurazone is not frequently used in burn centres in the United States.
Gentamicin is very effective against Gram-negative micro-organisms. Resistance to gentamicin may be developed and this resistance certainly limits the usage of this medication. Gentamicin has been shown to inhibit the activity of PMNs. Skin hypersensitivity has been reported with gentamicin [20].
Silver sulfadiazine (SSD) is a topical sulfonamide compound of silver nitrate and sodium sulfadiazine prepared as 1% water miscible cream. Silver sulfadiazine is effective against a wide range of flora, particularly Gram-negative bacteria like E. coli, Enterobacter, Klebsiella species, P. aeruginosa and Gram-positive bacteria like S. aureus and Candida albicans. 
Amongst the various topical antimicrobial agents available for the burn wounds, some of them specified above, sulphonamide derivatives have enjoyed a well deserved reputation in recent years for the treatment of different degrees of burn wounds infected with Gram-positive and Gram-negative bacterial infections as well as being effective against yeast. Amongst sulphonamides, the drug of choice for prophylaxis and treatment in most burn patients has been silver sulfadiazine (SSD). SSD is an effective broad spectrum antimicrobial agent commonly incorporated into topical creams used for burn wounds. In fact, since last five decades. SSD has been traditionally used for the treatment of different degrees of burn wounds infected with micro-organisms.
Past studies have shown that SSD inhibits bacteria that are resistant to other antimicrobial agents and that the compound is superior to many other antimicrobial agents. It acts on the cell membrane and cell wall of micro-organisms to produce its bactericidal effect. Silver is slowly released from the preparation in concentrations that are selectively toxic to bacteria. Silver also damages the DNA of the bacteria cell. Sulfadiazine, like other sulphonamides, inhibits bacterial synthesis of dihydrofolic acid by competing with para-aminobenzoic acid (PABA). It does not act on human cells.
Another antimicrobial agent namely chlorhexidine gluconate (CHG) is a powerful, relatively non-toxic antiseptic which has found widespread approval in current clinical practice [18]. CHG is an important antiseptic, disinfectant, antibacterial dental rinse, and preservative. It has wide antimicrobial spectrum and is effective against Gram-positive. Gram-negative bacteria, viruses and fungi. CHG binds to bacterial cell wall and cytoplasmic components leading to altered osmotic equilibrium and also precipitation of cytoplasmic components. At low concentrations, chlorhexidine is bacteriostatic; at higher concentrations, it is bactericidal. Chlorhexidine gluconate is a bisbiguanide that binds to the stratum corneum, providing sustained bactericidal and fungicidal activity. It does not lose its effectiveness in the presence of organic material, such as whole blood. CHG gets adsorbed onto the cell walls of microorganisms causing leakage of absorbing material from a wide variety of bacteria and affects the structure of proteins, inhibiting for example, membrane-bound ATPase [19], altering the configuration of proteins and facilitating the uptake of polymyxin by cells.
Though SSD is a standard treatment for burn wounds for the last fifty years, some studies showed that the compound delays the wound healing process, which is significant with second and third degree burns, due to delayed or less production of growth promoting factors using their receptors. Also, the absorption of silver from burn wounds led to silver toxicity leading to the impairment of dermal regeneration and decreased mechanical strength of dermal tissue. Several in vitro studies with human dermal fibroblasts have shown progressive cellular cytotoxicity with increasing concentrations of SSD and mafenide acetate. In the same studies the pre exposure of human dermal fibroblasts to EGF, basic fibroblast growth factor, or platelet-derived growth factor has resulted in cytoprotection of human dermal fibroblasts against effect of SSD [29].
Cases of bacterial resistance to silver have also been reported. Cason et al reported this in Gram-negative bacilli in burn wounds as early as 1966 [21]. In the late 1970s there were several reports of outbreaks of burn wound infection or colonisation by Gram-negative isolates resistant to SSD (Enterobacter cloacae, Providencia stuartii, and P. aeruginosa). It is clear that exposure to silver might select resistant micro-organisms and this could play important part in the predominance of intrinsically silver-resistant bacteria where silver is widely used.
Li et al reported the development of bacterial resistance to high concentration of silver (>1024 ppm) by repeated exposure to increasing concentrations in vitro [27].
The drawbacks related to the use of SSD for the treatment of burn wounds led to development of a formulation by Bharat Biotech International Limited (BBIL) comprising SSD with rh-EGF. This combination helps in the release of rh-EGF for a prolonged period and limitation of the delivery of silver necessary for optimum wound healing effect. The most important function of this SSD and rh-EGF combination is cytoprotective effect of rh-EGF against SSD and also helping in reversibility of the impaired burn wound healing process by the co-supplementation of EGF. The results established that this combination helps in reversal of cytotoxic effect of silver there by hastening wound healing process in burn patients. But concern has been raised regarding the potential for development of bacterial resistance against SSD and silver-resistant organisms reported in clinical samples due to permeability barrier.
Studies were done for the permeability of EGF into blood stream using rh-EGF of BBIL and SSD combination, and the results were found negative, which clearly showed that rh-EGF produced at BBIL doesn't enter into the blood stream. The combination of SSD with rh-EGF of BBIL trials in burn patients showed the effectiveness of the application of rh-EGF on the acceleration of the cicatrization process of dermal and hypodermal burns, resulting in a skin with an appearance, elasticity and colour identical to the normal skin and the major achievement being without hypertrophic scars. These evidences show the effect of rh-EGF on cicatrization, possibly due to its mitogenic effect.
Though the combination of rh-EGF and SSD had taken care of the delayed wound healing problem by reversal of SSD effect, the most important drawback in this formulation is that sulphonamide resistance is frequently noticed due to impermeable ability of SSD. Despite the medical benefits of using ionic silver to manage infections, concern has been raised regarding the potential for development of bacterial resistance and an association with cross-resistance to antibiotics has been implied. Silver-resistant organisms have been reported in clinical and environmental samples. The combination of SSD with EGF can only prevent the cytotoxic effect due to silver and helps in reversal of the impaired wound healing process. But this combination cannot be effective against the micro-organisms resistance against SSD due the permeability barrier. This combination is not sufficient in prevention of infection against the micro-organisms resistant to silver and this can lead to the cause of secondary infection which may prolong the wound healing process and also a serious threat to the burn wounds.
In the prior art a large number of antimicrobial agents have been tested and used. One of the most common agent among them have been the silver sulfadiazine (SSD) which has been used as alone or in combination with other antimicrobial agents like chlorhexidine gluconate (CHG) or a combination of SSD with rh-EGF as used by Bharat Biotech International Limited.
However, none of the prior art formulations were able to provide a much broader spectrum coverage, quicker wound healing without the risk of resistance development, metal ion toxicity, cytotoxicity etc.